Variations among Viruses in Influent Water and Effluent Water at a Wastewater Plant over One Year as Assessed by Quantitative PCR and Metagenomics.

Influent wastewater and effluent wastewater at the Rya treatment plant in Gothenburg, Sweden, were continuously monitored for enteric viruses by quantitative PCR (qPCR) during 1 year. Viruses in effluent wastewater were also identified by next-generation sequencing (NGS) in samples collected during spring, early summer, and winter. Samples of incoming wastewater were collected every second week. Seasonal variations in viral concentrations in incoming wastewater were found for noroviruses GII, sapovirus, rotavirus, parechovirus, and astrovirus. Norovirus GI and GIV and Aichi virus were present in various amounts during most weeks throughout the year, while hepatitis A virus, enterovirus, and adenovirus were identified less frequently. Fluctuations in viral concentrations in incoming wastewater were related to the number of diagnosed patients. The viruses were also detected in treated wastewater, however, with a 3- to 6-log10 reduction in concentration. Seven different hepatitis E virus (HEV) strains were identified in the effluents. Five of these strains belonged to genotype 3 and have been isolated in Sweden from swine, wild boars, and humans and in drinking water. The other two strains were divergent and had not been identified previously. They were similar to strains infecting rats and humans. Surveillance of enteric viruses in wastewater is a tool for early detection and follow-up of gastroenteritis outbreaks in society and for the identification of new viruses that can cause infection in humans.IMPORTANCE Both influent wastewater and treated wastewater at a wastewater treatment plant (WWTP) contain a high variety of human viral pathogens with seasonal variability when followed for 1 year. The peak of the amount of 11 different viruses in the inlet wastewater preceded the peak of the number of diagnosed patients by 2 to 4 weeks. The treatment of wastewater reduced viral concentrations by 3 to 6 log10 Despite the treatment of wastewater, up to 5 log10 virus particles per liter were released from into the surrounding river. Hepatitis E virus (HEV) strains previously identified in drinking water and two new strains, similar to those infecting rats and humans, were identified in the treated wastewater released from the WWTP.

Hepatitis E Virus Genotype 3 Genomes from RNA-Positive but Serologically Negative Plasma Donors Have CUG as the Start Codon for ORF3.

Hepatitis E virus (HEV) is a pathogen that causes hepatitis worldwide. Molecular studies have identified HEV RNA in blood products although its significance is not understood. This study was undertaken to characterize HEV genomes in asymptomatic plasma donors from Sweden and Germany lacking anti-HEV. Complete open reading frames (ORFs) were obtained from HEV strains in 5 out of 18 plasma donors who tested positive for HEV RNA. All strains had CUG as the start codon of ORF3, while 147 GenBank strains all had AUG as the start codon (p < 0.0001). This substitution was found in both interrelated and unrelated strains belonging to different phylogenetic clades. The HEV strains from the seronegative plasma donors had no other substitution in common, which may be why the CUG substitution seems to explain the seronegativity.

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes.

In order to improve genotyping and epidemiological analysis of Cryptosporidium spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different Cryptosporidium hominis patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encoding Cryptosporidium oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the Cryptosporidium parvum reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the C. hominis tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing Cryptosporidium directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of Cryptosporidium from human fecal samples, while alluding to the potential for a higher degree of genotyping within Cryptosporidium epidemiology.

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana.

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.

Building a precision medicine infrastructure at a national level: The Swedish experience.

Precision medicine has the potential to transform healthcare by moving from one-size-fits-all to personalised treatment and care. This transition has been greatly facilitated through new high-throughput sequencing technologies that can provide the unique molecular profile of each individual patient, along with the rapid development of targeted therapies directed to the Achilles heels of each disease. To implement precision medicine approaches in healthcare, many countries have adopted national strategies and initiated genomic/precision medicine initiatives to provide equal access to all citizens. In other countries, such as Sweden, this has proven more difficult due to regionally organised healthcare. Using a bottom-up approach, key stakeholders from academia, healthcare, industry and patient organisations joined forces and formed Genomic Medicine Sweden (GMS), a national infrastructure for the implementation of precision medicine across the country. To achieve this, Genomic Medicine Centres have been established to provide regionally distributed genomic services, and a national informatics infrastructure has been built to allow secure data handling and sharing. GMS has a broad scope focusing on rare diseases, cancer, pharmacogenomics, infectious diseases and complex diseases, while also providing expertise in informatics, ethical and legal issues, health economy, industry collaboration and education. In this review, we summarise our experience in building a national infrastructure for precision medicine. We also provide key examples how precision medicine already has been successfully implemented within our focus areas. Finally, we bring up challenges and opportunities associated with precision medicine implementation, the importance of international collaboration, as well as the future perspective in the field of precision medicine.

Self-reported symptom severity, general health, and impairment in post-acute phases of COVID-19: retrospective cohort study of Swedish public employees.

This study aimed to examine current symptom severity and general health in a sample of primarily non-hospitalized persons with polymerase chain reaction (PCR) confirmed COVID-19 in comparison to PCR negative controls. During the first quarter of 2021, we conducted an online survey among public employees in West Sweden, with a valid COVID-19 test result. The survey assessed past-month severity of 28 symptoms and signs, self-rated health, the WHO Disability Assessment Schedule (WHODAS) 2.0 and illness severity at the time of test. We linked participants' responses to their SARS-CoV-2 PCR tests results. We compared COVID-19 positive and negative participants using univariable and multivariable regression analyses. Out of 56,221 invited, 14,222 (25.3%) responded, with a response rate of 50% among SARS-CoV-2 positive individuals. Analysis included 10,194 participants (86.4% women, mean age 45 years) who tested positive 4-12 weeks (N = 1425; subacute) and > 12 weeks (N = 1584; postcovid) prior to the survey, and 7185 PCR negative participants who did not believe that they had had COVID-19. Symptoms were highly prevalent in all groups, with worst symptoms in subacute phase participants, followed by postcovid phase and PCR negative participants. The most specific symptom for COVID-19 was loss of smell or taste. Both WHODAS 2.0 score and self-rated health were worst in subacute participants, and modestly worse in postcovid participants than in negative controls. Female gender, older age and acute illness severity had larger effects on self-rated health and WHODAS 2.0 score in PCR positive participants than in PCR negative. Studies with longer follow-up are needed to determine the long-term improvement after COVID-19.

Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics.

Infection in the central nervous system is a severe condition associated with high morbidity and mortality. Despite ample testing, the majority of encephalitis and meningitis cases remain undiagnosed. Metagenomic sequencing of cerebrospinal fluid has emerged as an unbiased approach to identify rare microbes and novel pathogens. However, several major hurdles remain, including establishment of individual limits of detection, removal of false positives and implementation of universal controls. Twenty-one cerebrospinal fluid samples, in which a known pathogen had been positively identified by available clinical techniques, were subjected to metagenomic DNA sequencing. Fourteen samples contained minute levels of Epstein-Barr virus. The detection threshold for each sample was calculated by using the total leukocyte content in the sample and environmental contaminants found in the bioinformatic classifiers. Virus sequences were detected in all ten samples, in which more than one read was expected according to the calculations. Conversely, no viral reads were detected in seven out of eight samples, in which less than one read was expected according to the calculations. False positive pathogens of computational or environmental origin were readily identified, by using a commonly available cell control. For bacteria, additional filters including a comparison between classifiers removed the remaining false positives and alleviated pathogen identification. Here we show a generalizable method for identification of pathogen species using DNA metagenomic sequencing. The choice of bioinformatic method mainly affected the efficiency of pathogen identification, but not the sensitivity of detection. Identification of pathogens requires multiple filtering steps including read distribution, sequence diversity and complementary verification of pathogen reads.

Comparative Genomics of Clinical Isolates of the Emerging Tick-Borne Pathogen Neoehrlichia mikurensis.

Tick-borne 'Neoehrlichia (N.) mikurensis' is the cause of neoehrlichiosis, an infectious vasculitis of humans. This strict intracellular pathogen is a member of the family Anaplasmataceae and has been unculturable until recently. The only available genetic data on this new pathogen are six partially sequenced housekeeping genes. The aim of this study was to advance the knowledge regarding 'N. mikurensis' genomic relatedness with other Anaplasmataceae members, intra-species genotypic variability and potential virulence factors explaining its tropism for vascular endothelium. Here, we present the de novo whole-genome sequences of three 'N. mikurensis' strains derived from Swedish patients diagnosed with neoehrlichiosis. The genomes were obtained by extraction of DNA from patient plasma, library preparation using 10× Chromium technology, and sequencing by Illumina Hiseq-4500. 'N. mikurensis' was found to have the next smallest genome of the Anaplasmataceae family (1.1 Mbp with 27% GC contents) consisting of 845 protein-coding genes, every third of which with unknown function. Comparative genomic analyses revealed that 'N. mikurensis' was more closely related to Ehrlichia chaffeensis than to Ehrlichia ruminantium, the opposite of what 16SrRNA sequence-based phylogenetic analyses determined. The genetic variability of the three whole-genome-sequenced 'N. mikurensis' strains was extremely low, between 0.14 and 0.22‰, a variation that was associated with geographic origin. No protein-coding genes exclusively shared by N. mikurensis and E. ruminantium were identified to explain their common tropism for vascular endothelium.

Development and characterization of an oat TILLING-population and identification of mutations in lignin and beta-glucan biosynthesis genes.

BACKGROUND: Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved beta-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda. RESULTS: Here a population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots were obtained. The M2 population was initially evaluated by visual inspection and a number of different phenotypes were seen ranging from dwarfs to giants, early flowering to late flowering, leaf morphology and chlorosis. Phloroglucinol/HCl staining of M3 seeds, obtained from 1824 different M2 lines, revealed a number of potential lignin mutants. These were later confirmed by quantitative analysis. Genomic DNA was prepared from the M2 population and the mutation frequency was determined. The estimated mutation frequency was one mutation per 20 kb by RAPD-PCR fingerprinting, one mutation per 38 kb by MALDI-TOF analysis and one mutation per 22.4 kb by DNA sequencing. Thus, the overall mutation frequency in the population is estimated to be one mutation per 20-40 kb, depending on if the method used addressed the whole genome or specific genes. During the investigation, 6 different mutations in the phenylalanine ammonia-lyase (AsPAL1) gene and 10 different mutations in the cellulose synthase-like (AsCslF6) beta-glucan biosynthesis gene were identified. CONCLUSION: The oat TILLING population produced in this work carries, on average, hundreds of mutations in every individual gene in the genome. It will therefore be an important resource in the development of oat with specific characters. The population (M5) will be available for academic research via Nordgen http://www.nordgen.org as soon as enough seeds are obtained.[Genbank accession number for the cloned AsPAL1 is GQ373155 and GQ379900 for AsCslF6].

Hepatitis E virus genotype 3 strains and a plethora of other viruses detected in raw and still in tap water.

In this study, next generation sequencing was used to explore the virome in 20L up to 10,000L water from different purification steps at two Swedish drinking water treatment plants (DWTPs), and in tap water. One DWTP used ultrafiltration (UF) with 20 nm pores, the other UV light treatment after conventional treatment of the water. Viruses belonging to 26 different families were detected in raw water, in which 6-9 times more sequence reads were found for phages than for known environmental, plant or vertebrate viruses. The total number of viral reads was reduced more than 4-log10 after UF and 3-log10 over UV treatment. However, for some viruses the reduction was 3.5-log10 after UF, as for hepatitis E virus (HEV), which was also detected in tap water, with sequences similar to those in raw water and after treatment. This indicates that HEV had passed through the treatment and entered into the supply network. However, the viability of the viruses is unknown. In tap water 10-130 International Units of HEV RNA/mL were identified, which is a comparable low amount of virus. The risk of getting infected through consumption of tap water is probably negligible, but needs to be investigated. The HEV strains in the waters belonged to subtypes HEV3a and HEV3c/i, which is associated with unknown source of infection in humans infected in Sweden. None of these subtypes are common among pigs or wild boar, the major reservoirs for HEV, indicating that water may play a role in transmitting this virus. The results indicate that monitoring small fecal/oral transmitted viruses in DWTPs may be considered, especially during community outbreaks, to prevent potential transmission by tap water.

ß-Mannosidosis caused by a novel homozygous intragenic inverted duplication in MANBA.

ß-Mannosidosis is a lysosomal storage disorder characterized by accumulation of disaccharides due to deficiency of the lysosomal enzyme ß-mannosidase. The disease is caused by mutations in MANBA and is extremely rare in humans. Although the clinical presentation is heterogeneous, common symptoms include various degrees of developmental delay, behavioral disturbances, hearing loss, and frequent infections. We report a 15-yr-old girl presenting with mild intellectual disability, sensorineural hearing loss, severe behavioral disturbances, dysmorphic traits, and evolving angiokeratomas. Copy-number variation analysis of next-generation sequencing (NGS) data indicated increased coverage in exons 8-11 of MANBA Low ß-mannosidase activity (1 µkatal/kg protein, refv 25-40) established the diagnosis of ß-mannosidosis. Whole-genome sequencing (WGS) and cDNA analysis revealed a novel homozygous intragenic inverted duplication in MANBA, where a 13.1-kb region between introns 7 and 11 was duplicated and inserted in an inverted orientation, creating a 67-base nonduplicated gap at the insertion point. Both junctions showed microhomology regions. The inverted duplication resulted in exon skipping of exons 8-9 or 8-10. Our report highlights the importance of copy-number variation analysis of data from NGS and in particular the power of WGS in the identification and characterization of copy-number variants.

γ-glutamyl transpeptidase deficiency caused by a large homozygous intragenic deletion in GGT1.

γ-Glutamyl transpeptidase deficiency (glutathionuria, OMIM 231950) is a rare disease, with only six patients reported in the literature, although this condition has probably been underdiagnosed due the difficulty to routinely analyze glutathione in clinical samples and to the fact that no genetic defect has been coupled to the disease so far. We report two siblings with mild psychomotor developmental delay and mild neurological symptoms, who presented a markedly increased excretion of glutathione in urine and a very low γ-glutamyl transpeptidase activity in serum. Whole-genome sequencing revealed the presence of a 16.9 kb homozygous deletion in GGT1, one of the genes encoding enzymes with γ-glutamyl transpeptidase activity in the human genome. Close analysis revealed the presence of a 13 bp insertion at the deletion junction. This is the first report of a genetic variant as the cause of glutathionuria. In addition, genetic characterization of the patients' parents and a healthy sibling has provided direct genetic evidence regarding the autosomal recessive nature of this disease.

Differential removal of human pathogenic viruses from sewage by conventional and ozone treatments.

Sewage contains a mixed ecosystem of diverse sets of microorganisms, including human pathogenic viruses. Little is known about how conventional as well as advanced treatments of sewage, such as ozonation, reduce the environmental spread of viruses. Analyses for viruses were therefore conducted for three weeks in influent, after conventional treatment, after additional ozonation, and after passing an open dam system at a full-scale treatment plant in Knivsta, Sweden. Viruses were concentrated by adsorption to a positively charged filter, from which they were eluted and pelleted by ultracentrifugation, with a recovery of about 10%. Ion Torrent sequencing was used to analyze influent, leading to the identification of at least 327 viral species, most of which belonged to 25 families with some having unclear classification. Real-time PCR was used to test for 21 human-related viruses in inlet, conventionally treated, and ozone-treated sewage and outlet waters. The viruses identified in influent and further analyzed were adenovirus, norovirus, sapovirus, parechovirus, hepatitis E virus, astrovirus, pecovirus, picobirnavirus, parvovirus, and gokushovirus. Conventional treatment reduced viral concentrations by one to four log10, with the exception of adenovirus and parvovirus, for which the removal was less efficient. Ozone treatment led to a further reduction by one to two log10, but less for adenovirus. This study showed that the amount of all viruses was reduced by conventional sewage treatment. Further ozonation reduced the amounts of several viruses to undetectable levels, indicating that this is a promising technique for reducing the transmission of many pathogenic human viruses.

[New subtype of molluscipoxvirus detected]. / Ny subtyp av molluscipoxvirus påvisad - Modern teknik identifierar nya och ovanliga patogener snabbare.

Molluscum contagiosum is a viral infection of the epidermis characterized by skin-colored papules or nodules frequently with a central depression. Atypical variants may occur, primarily in immunosuppressed individuals. We here report a case of ¼giant Molluscum contagiosum« in an immunocompetent child. The patient was presented with a fairly smooth nodule of 2 cm in diameter on the ring finger. Molluscipoxvirus-like virus particles were detected by electron microscopy from the nodule, but since the clinical picture was not compatible with MC, next generation sequencing was performed in order to verify the diagnosis.  Of the total number of obtained sequences, 25% belonged to molluscipoxvirus (MCV) and de novo assembly revealed three contigs corresponding to 95% of the MCV genome. The assembled genome was compared to previously published sequences of the ¼major envelope protein« used for genotyping of MCV genus. Several unique single nucleotide polymorphisms were identified, which led us to classify this virus as a new subtype of MCV.

Draft Genome Sequence of Moraxella catarrhalis Type Strain CCUG 353T.

Moraxella catarrhalis is a Gram-negative commensal and pathogenic bacterium found in the human respiratory tract. It is associated with otitis media and respiratory tract infections. Here, we report the draft genome sequence of M. catarrhalis type strain CCUG 353(T), composed of 18 contigs and a total size of 1.89 Mb.

Limited clonal relatedness between gut IgA plasma cells and memory B cells after oral immunization.

Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8(high)/GFP(+) NP-specific B cells. Unexpectedly, memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4ß7-integrin(+)CD73(+)PD-L2(+)CD80(+) and at systemic sites mostly IgM(+), while 80% are IgA(+) in Peyer's patches. On reactivation, most memory B cells in Peyer's patches are GL7(-), but expand in germinal centres and acquire higher affinity and more mutations, demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus, gut mucosal memory possesses unique features not seen after systemic immunization.

Draft Genome Sequence of Streptococcus gordonii Type Strain CCUG 33482T.

Streptococcus gordoniitype strain CCUG 33482(T)is a member of theStreptococcus mitisgroup, isolated from a case of subacute bacterial endocarditis. Here, we report the draft genome sequence ofS. gordoniiCCUG 33482(T), composed of 41 contigs of a total size of 2.15 Mb with 2,061 annotated coding sequences.

Comparative Transcriptomics of Sijung and Jumli Marshi Rice during Early Chilling Stress Imply Multiple Protective Mechanisms.

INTRODUCTION: Low temperature is one of the major environmental factors that adversely affect plant growth and yield. Many cereal crops from tropical regions, such as rice, are chilling sensitive and, therefore, are affected already at <10 °C. Interestingly, it has been demonstrated that chilling susceptibility varies greatly among rice varieties, which indicates differences in the underlying molecular responses. Understanding these differences is vital for continued development of rational breeding and transgenic strategies for more tolerant varieties. Thus, in this study, we conducted a comparative global gene expression profiling analysis of the chilling tolerant varieties Sijung and Jumli Marshi (spp. Japonica) during early chilling stress (<24 h, 10 °C). METHODS AND RESULTS: Global gene expression experiments were conducted with Agilent Rice Gene Expression Microarray 4 x 44 K. The analysed results showed that there was a relatively low (percentage or number) overlap in differentially expressed genes in the two varieties and that substantially more genes were up-regulated in Jumli Marshi than in Sijung but the number of down-regulated genes were higher in Sijung. In broad GO annotation terms, the activated response pathways in Sijung and Jumli Marshi were coherent, as a majority of the genes belonged to the catalytic, transcription regulator or transporter activity categories. However, a more detailed analysis revealed essential differences. For example, in Sijung, activation of calcium and phosphorylation signaling pathways, as well as of lipid transporters and exocytosis-related proteins take place very early in the stress response. Such responses can be coupled to processes aimed at strengthening the cell wall and plasma membrane against disruption. On the contrary, in Jumli Marshi, sugar production, detoxification, ROS scavenging, protection of chloroplast translation, and plausibly the activation of the jasmonic acid pathway were the very first response activities. These can instead be coupled to detoxification processes. CONCLUSIONS: Based on the results inferred from this study, we conclude that different, but overlapping, strategies are undertaken by the two varieties to cope with the chilling stress; in Sijung the initial molecular responses seem to be mainly targeted at strengthening the cell wall and plasma membrane, whereas in Jumli Marshi the protection of chloroplast translation and detoxification is prioritized.